The Tol-Pal System of Uropathogenic Escherichia coli Is Responsible for Optimal Internalization Into and Aggregation Within Bladder Epithelial Cells, Colonization of the Urinary Tract of Mice, and Bacterial Motility

Abstract

Urinary tracts infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is a common infectious disease. With the shortage of new antimicrobial agents, the increase in UPEC resistance to commonly used drugs, such as fluoroquinolones and β-lactams including carbapenems is a critical issue. UPEC invades urinary tract cells, where it aggregates, and subsequently, forms biofilm-like multicellular colonies termed intracellular bacterial communities (IBCs). This process allows the bacteria to establish infections and so may be a good potential target for new drugs to treat infections. Here, we show that deletion of the tolB gene, encoding a protein of the Tol-Pal system that was originally characterized as a protein complex for colicin uptake and maintenance of the outer membrane, decreases the level of bacterial internalization into and aggregation within cultured bladder epithelial cells and also inhibits the colonization of mice urinary tracts. The tolB mutant also exhibited defective motility because of impaired flagellum syntheses. The fliC and motA mutants, which are non-motile strains, also exhibited lower levels of bacterial internalization and aggregation than their wild-type parent. Additional deletion of tolB in the fliC mutant did not further decrease these, suggesting that the attenuated virulence of the tolB mutant is a result of defective motility. The tolA , tolQ , tolR , and pal mutants that lack other members of the Tol-Pal system also exhibited lower levels of motility and aggregation within bladder epithelial cells compared to their wild-type parent. These combined results suggest another role of the Tol-Pal system, i.e., that it is responsible for optimal internalization, aggregation followed by IBC formation within urinary tract cells, and bacterial motility.

Roles of the Tol-Pal system in the Type III secretion system and flagella-mediated virulence in enterohemorrhagic Escherichia coli

“…The disturbance in the outer membrane by deletion of tol - pal genes may impair the precise localization and stabilization of the outer ring, which presumably leads to disassembly of the transport protein complex. However, some proteins localized in the outer membrane, such as fimbrial protein complexes, remain functional in the tol - pal mutants despite the absence of activity of the Tol-Pal system 21 . It would be interesting to gain insight into which proteins are affected by the Tol-Pal system in addition to Type III transport proteins and how these proteins are associated with it.…”

“…Bacteria were cultured for 24 h at 30 °C in Heart Infusion medium containing 1.5% agar. Flagella were stained with Victoria blue/tannic acid solution as described previously 21 .…”

“…Total RNA extraction and cDNA synthesis were performed using a SV Total-RNA Isolation System (Promega Corp., Madison, WI, United States) and ReverTra Ace qPCR RT Manster Mix (TOYOBO Co. Ltd, Osaka, Japan), respectively, following the manufacturer’s instructions. Real-time PCR were carried out as described previously 21 . Primers were listed in Table 4 .…”

“…Bacterial adhesion to HeLa cells were assessed as s described previously with slight modifications 21 . Bacteria were inoculated into HeLa cells in 24-well plates.…”

“…Some of tol - pal genes are also involved in bacterial pathogenesis, such as survival of Salmonella enterica serovar Typhimurium within macrophages, cytotoxicity of Erwinia chrysanthemi in plant cells, and pustule formation in humans by Haemophilus ducreyi 15 , 17 – 20 . Previously, we found that mutants in tol - pal genes of uropathogenic E. coli (UPEC) exhibited decreased bacterial internalization in urinary tract cells and impaired motility, which reduced bacterial colonization within the urinary tract of mice 21 . Thus, the Tol-Pal system contributes to the pathogenicity of UPEC in the urinary tract.…”